Project Summary Mycobacterium tuberculosis (Mtb) has adapted to survive a wide range of assaults?from our immune response to antimicrobial therapeutics?intended to eradicate the organism. However, the molecular switches that enable Mtb to endure these stresses, to slow replication or to become dormant as a latent tuberculosis infection (LTBI) are not known. Emerging studies on the molecular underpinnings of stress survival in Escherichia coli generally point to a major role for toxin-antitoxin (TA) systems, which are operons comprising adjacent genes encoding two small proteins, a toxin and its cognate antitoxin that inhibits toxin activity in the TA protein-protein complex. Their expression has been implicated in Mtb stress survival and/or the switch to the non-replicating persistent state characteristic of LTBI. However, several bottlenecks have impeded progress toward rigorous testing of this provocative association. This proposal enlists a proven collaborative team with combined expertise in four core components of the proposed work--toxin-antitoxin systems, M. tuberculosis biology and physiology, bioinformatics/computational biology and RNA-seq. The goal of this R21 proposal is to enlist our 5? RNA-seq technology to identify the RNA targets among the uncharacterized Mtb VapC toxin family members. This approach directly tests our hypothesis that tRNA-cleaving VapC toxins collude to downregulate Mtb growth by surgically inactivating a single tRNA, followed by translation inhibition of key proteins that contribute to growth regulation that require this depleted tRNA. In addition to illuminating the mechanism of action of the VapC family of toxins, these studies should uncover new insights into the full spectrum of molecular events that underlie the growth regulating properties of these toxins. A better understanding of these processes is essential for the development of more effective treatments for LTBI. !